ArticlePDF Available

7T-MRI of Transferrin Receptor and Ferritin Gene Expression in a Mouse Neural Stem Cell Line

Authors:

Abstract

Introduction and Background. The possibility of utilizing endogenous iron as an MRI contrast agent has emerged with the development of high magnetic field imaging due to its increased sensitivity to the paramagnetic ion. By manipulating the genes involved in normal iron regulation, causing a cell to increase uptake and stable storage of iron, it has been proposed that these genes could be used as an MRI reporter of associated gene expression. In previous literature, the transferrin receptor [1] and ferritin [2] have individually been overexpressed in in vivo systems and were found to lead to either insufficient iron accumulation for MRI visualization or cellular toxicity. In this study, we attempted to boost iron accumulation to an MRI detectable level by over-expression of both the transferrin receptor and ferritin proteins in an in vitro system. Materials and Methods. Cell lines: The C17 mouse cerebellar progenitor cell line [3] was transfected with a construct consisting of the human transferrin receptor (hTfR) and the human ferritin H chain (hFTH) genes connected by an internal ribosomal entry site (IRES) in a pZeo-SV2(+) vector (Invitrogen) [4]. The construct was transfected by electroporation and zeocin-resistant subclones were selected. Based on RT-PCR confirmation of expression of both genes, subclone #12 was selected for further analysis. Transferrin/Iron supplementation and sample preparation: The C17 control and #12 subclone were grown in standard growth medium (DMEM with 10% FBS, 5% Horse serum, 200mM L-glutamine, 100mM sodium pyruvate) or in standard medium supplemented with human holo-transferrin (1mg/mL) and iron citrate (1mM) for 48 hours prior to imaging and histology. For imaging, the cells were washed to remove unbound iron, pelleted by low rpm centrifugation into glass NMR tubes and mounted in an imaging phantom. For histology, cells were washed and fixed in 4% PFA for 10 minutes prior to PPB staining and neutral red counterstain. MRI and data analysis: Imaging was performed on a SMIS console interfaced to a 7T horizontal bore magnet with 250mT/m actively shielded gradients (Magnex) using a quadrature birdcage coil (ID=35mm, L=46mm) and the following parameters: T1 (3DGE, Mx: 512x256x256, FOV: 80x40x40, TR/TE= 50/5ms, FA=65º), T2 (2DSE, Mx: 512x512, FOV: 40x40, TR/TE= 2000/50ms, slice thickness= 250µM), and T2* (3DGE, Mx and FOV as in T1, TR/TE=50/15ms, FA=20º). The mean signal intensity was obtained by manual selection of ROIs corresponding to each cell pellet in a cross sectional image and SMIS system measurement. Results. Iron staining shows grossly increased iron accumulation (blue precipitate) in the #12 subclone when grown in supplemented conditions versus C17 control cells (Fig.1a). T1 images showed no signal difference between cell types with or without supplementation (data not shown). However, both T2 and T2*-weighted images showed increased signal loss in the #12 subclone with supplementation versus the supplemented C17 control (Fig. 1b, c). T2 mean signal intensity decreased by a mean of 70% in supplemented #12 cell pellets versus 58% in C17 controls with supplementation (Fig. 2a). T2* mean signal intensity decreased in the #12 subclone by 61% with iron supplementation while C17 control signal decreased by 39% with supplementation (Fig 2b). Initial relaxometry on similar fixed samples showed a decrease in T2 with iron supplementation in the #12 subclone of 63% (from 129ms to 48ms) versus 45% in C17 controls (from 125ms to 68ms). Conclusions. These results indicate that cells overexpressing transferrin receptor and ferritin proteins in combination can accumulate sufficient iron for visualization by MRI, showing the potential of this approach for controlling cell-specific MR contrast.
A preview of the PDF is not available
Article
With the enormous and growing number of experimental and genetic mouse models of human disease, there is a need for efficient means of characterizing abnormalities in mouse anatomy and physiology. Adaptation of magnetic resonance imaging (MRI) to the scale of the mouse promises to address this challenge and make major contributions to biomedical research by non-invasive assessment in the mouse. MRI is already emerging as an enabling technology providing informative and meaningful measures in a range of mouse models. In this review, recent progress in both in vivo and post mortem imaging is reported. Challenges unique to mouse MRI are also identified. In particular, the needs for high-throughput imaging and comparative anatomical analyses in large biological studies are described and current efforts at handling these issues are presented.
Article
Mouse models are crucial for the study of genetic factors and processes that influence human disease. In addition to tools for measuring genetic expression and establishing genotype, tools to accurately and comparatively assess mouse phenotype are essential in order to characterize pathology and make comparisons with human disease. MRI provides a powerful means of evaluating various anatomical and functional changes and hence is growing in popularity as a phenotypic readout for biomedical research studies. To accommodate the large numbers of mice needed in most biological studies, mouse MRI must offer high-throughput image acquisition and efficient image analysis. This article reviews the technology of multiple-mouse MRI, a method that images multiple mice or specimens simultaneously as a means of enabling high-throughput studies. Aspects of image acquisition and computational analysis in multiple-mouse studies are also described.
ResearchGate has not been able to resolve any references for this publication.